The invention is directed to mutant luciferase enzymes having greatly increased thermostability compared to natural luciferases or to luciferases from which they are derived as measured, e.g., by half-lives of at least 2 hours at 50xc2x0 C. in aqueous solution. The invention includes mutant luciferase enzymes that are resistant to inhibition by a substrate inhibitor, e.g., a substrate analog. The invention is also drawn to polynucleotides encoding the novel luciferases, and to hosts transformed to express the luciferases. The invention is further drawn to methods of producing luciferases with increased thermostability and the use of these luciferases in any method in which previously known luciferases are conventionally employed. Some of the uses employ kits. The invention also provides a method of producing a polynucleotide sequence encoding an enzyme that is resistant to inhibition by an inhibitor, and a method which yields a polynucleotide sequence encoding an enzyme having enhanced enzymological properties.
Luciferases are defined by their ability to produce luminescence. Beetle luciferases form a distinct class with unique evolutionary origins and chemical mechanisms (Wood, 1995).
Although the enzymes known as beetle luciferases are widely recognized for their use in highly sensitive luminescent assays, their general utility has been limited due to low thermostability. Beetle luciferases having amino acid sequences encoded by cDNA sequences cloned from luminous beetles are not stable even at moderate temperatures. For example, even the most stable of the luciferases, LucPpe2, obtained from a firefly has very little stability at the moderate temperature of 37xc2x0 C. Firefly luciferases are a sub-group of the beetle luciferases. Historically, the term xe2x80x9cfirefly luciferasexe2x80x9d referred to the enzyme LucPpy from a single species Photinus pyralis (Luc+is a mutant version of LucPpy, see U.S. Pat. No. 5,670,356).
Attempts have been reported to mutate natural cDNA sequences encoding luciferase and to select mutants for improved thermostability (White et al., 1994; from P. pyralis, and Kajiyama and Nekano, 1993; from Luciola lateralis.) However, there is still a need to improve the characteristics and versatility of this important class of enzymes.
The invention is drawn to novel and remarkably thermostable luciferases, including luciferase enzymes with half-lives of at least 2 hours at 50xc2x0 C., or at least 5 hours at 50xc2x0 C., in an aqueous solution. As described hereinbelow, after 2 hours at 50xc2x0 C. in an aqueous solution, a thermostable luciferase of the invention lost less than 5% luminescence activity. The mutant luciferases of the present invention display remarkable and heretofore unrealized thermostability at 22xc2x0 C. in an aqueous solution and at temperatures at least as high as 60xc2x0 C. in an aqueous solution. For example, the luciferases of the invention are thermostable for at least 10 hours at 50xc2x0 C.; for at least 2 hours, preferably at least 5 hours, more preferably at least 10 hours, and even more preferably at least 24 hours, at 60xc2x0 C.; and/or for at least 100 days, preferably at least 200 days, more preferably at least 500 days, and even more preferably at least 800 days, at 22xc2x0 C., in aqueous solution. For example, after 30 days at 22xc2x0 C. in an aqueous solution, a thermostable luciferase of the invention lost less than 5% luminescence activity. Preferably, the thermostable luciferases of the invention have enhanced luminescence intensity, enhanced signal stability, enhanced substrate utilization, and/or decreased Km, relative to a reference, e.g., a native wild-type, luciferase. The invention is further directed to the mutant luciferase genes (e.g., cDNA or RNA) which encode the novel luciferase enzymes. The terminology used herein is, e.g., for the mutants isolated in experiment 90, plate number 1, well B5, the E. coli strain is 90-1B5, the mutant gene is luc90-1B5, and the mutated luciferase is Luc90-1B5.
As defined herein, a xe2x80x9cthermostablexe2x80x9d enzyme, e.g., a luciferase, or an enzyme which has xe2x80x9cthermostabilityxe2x80x9d, is an enzyme which under certain conditions, e.g., at certain temperature, in aqueous solution and/or for certain periods of time, has an increased retention of activity relative to a reference enzyme. For example, for a thermostable luciferase, a reference luciferase may be native wild-type luciferase or recombinant wild-type luciferase. Preferably, for beetle luciferases, the activity is luminescence under conditions of saturation with luciferin and ATP. One measure of thermostability of an enzyme is the half-life of the enzyme in an aqueous solution (the time over which 50% of the activity is lost) at a stated temperature.
The invention further encompasses expression vectors and other genetic constructs containing the mutant luciferases, as well as hosts, bacterial and otherwise, transformed to express the mutant luciferases. The invention is also drawn to compositions and kits which contain the novel luciferases, and use of these luciferases in any methodology where luciferases are employed.
Various means of random mutagenesis were applied to a luciferase gene (nucleotide sequence), most particularly gene synthesis using an error-prone polymerase, to create libraries of modified luciferase genes. This library was expressed in colonies of E. coli and visually screened for efficient luminescence to select a subset library of modified luciferases. Lysates of these E. coli strains were then made, and quantitatively measured for luciferase activity and thermostability. From this, a smaller subset of modified luciferases was chosen, and the selected mutations were combined to make composite modified luciferases. New libraries were made from the composite modified luciferases by random mutagenesis and the process was repeated. The luciferases with the best overall performance were selected after several cycles of this process.
Methods of producing improved luciferases include directed evolution using a polynucleotide sequence encoding a first beetle luciferase as a starting (parent) sequence, to produce a polynucleotide sequence encoding a second luciferase with increased thermostability, compared to the first luciferase, while maintaining other characteristics of the enzymes. A cDNA designated lucPpe2 encodes a firefly luciferase derived from Photuris pennsylvanica that displays increased thermostability as compared to the widely utilized luciferase designated LucPpy from Photinus pyralis. The cDNA encoding LucPpe2 was isolated, sequenced and cloned (see Leach et al., 1997). A mutant of this gene encodes a first luciferase LucPpe2 [T249M]. However, the methods of the invention are not limited to use with a polynucleotide sequence encoding a beetle luciferase, i.e., the methods of the invention may be employed with a polynucleotide sequence encoding other enzymes.
In an embodiment of a mutant luciferase, the amino acid sequence is that of LucPpe2 shown in FIG. 45 with the exception that at residue 249 there is a M (designated T249M) rather than the T reported by Leach et al. The underlined residue (249) shows mutation from T to M. This enzyme produced approximately 5-fold more light in vivo when expressed in E. coli. 
Diluted extracts of recombinant E. coli that expressed mutant luciferases made by the methods of the invention were simultaneously screened for a plurality of characteristics including light intensity, signal stability, substrate utilization (Km), and thermostability. A fully automated robotic system was used to screen large numbers of mutants in each generation of the evolution. After several cycles of mutagenesis and screening, thereby creating mutant libraries of luciferases, an increased thermostability compared to LucPpe2 [T249M] of about 35xc2x0 C. was achieved for clone Luc90-1B5 which also essentially maintained enzymatic activity (there was only negligible loss in activity of 5%) when kept in aqueous solution over 2 hours at 50xc2x0 C., 5 hours at 65xc2x0 C., or over 6 weeks at 22xc2x0 C.
Mutant luciferases of the present invention display increased thermostability for at least 2 hours at 50xc2x0 C., preferably at least 5 hours at 50xc2x0 C., and in the range of at least 2 hours, preferably at least 24 hours, and more preferably at least 50 hours, at temperatures including 50xc2x0 C., 60xc2x0 C., and/or at temperatures up to 65xc2x0 C. In particular, the present invention comprises thermostable mutant luciferases which, when solubilized in a suitable aqueous solution, have a thermostability greater than about 2 hours at about 50xc2x0 C., more preferably greater than about 10 hours at 50xc2x0 C., and more preferably still greater than 5 hours at 50xc2x0 C. The present invention also comprises mutant luciferases which, when solubilized in a suitable aqueous solution, have a thermostability greater than about 2 hours, more preferably at least 5 hours, even more preferably greater than about 10 hours, and even more preferably still greater than about 24 hours, at about 60xc2x0 C. The present invention further comprises mutant luciferases which when solubilized in a suitable aqueous solution have a thermostability greater than about 3 months at about 22xc2x0 C., and more preferably a thermostability of at least 6 months at 22xc2x0 C. An embodiment of the invention is a luciferase mutant having thermostability at 65xc2x0 C., wherein a loss of activity of about 5-6% was found after 6 hours (equivalent to a half-life of 2 days). The half-lives of enzymes from the most stable clones of the present invention, extrapolated from data showing small relative changes, is greater than 2 days at 65xc2x0 C. (corresponding to 6% loss over 6 hours), and about 2 years at 22xc2x0 C. (corresponding to 5% loss over 9 weeks).
In particular, the invention comprises luciferase enzymes with embodiments of amino acid sequences disclosed herein (e.g., mutant luciferases designated Luc49-7C6, Luc78-0B10; Luc90-1B5, Luc133-1B2, and Luc146-1H2, as well as all other beetle luciferases that have thermostability as measured in half-lives of at least 2 hours at 50xc2x0 C. The invention also comprises mutated polynucleotide sequences encoding luciferase enzymes containing any single mutation or any combination of mutations of the type which convert an amino acid of the reference beetle luciferase into a consensus amino acid. Conserved amino acids are defined as those that occur at a particular position in all sequences in a given set of related enzymes. Consensus amino acids are defined as those that occur at a particular position in more than 50% of the sequences in a given set of enzymes. An example is the set of beetle luciferase sequences shown in FIG. 19, excluding LucPpe2.
Nucleotide sequences encoding beetle luciferases are aligned in FIG. 19. Eleven sequences found in nature in various genera, and species within genera, are aligned, including lucPpe2. There are at least three mutations present in each mutant luciferase that show increased thermostability. In general, mutations are not of a conserved amino acid residue. The mutations in the mutant luciferases are indicated in FIGS. 22-47 by underlining.
The invention also provides methods to prepare enzymes having one or more desired properties, e.g., resistance to inhibition by a substrate analog of the enzyme or enhanced enzymological properties. The method comprises selecting at least one isolated polynucleotide sequence encoding an enzyme with the desired property, e.g., an enzymological property, from a first population of mutated polynucleotide sequences. The selected, isolated polynucleotide sequence is then mutated to yield a second population of mutated polynucleotide sequences. Preferably, a mixture of selected isolated polynucleotide sequences are mutated to yield a second population of mutated polynucleotide sequences. The process may be repeated until a further polynucleotide sequence is obtained, e.g., selected and/or isolated, which further polynucleotide sequence encodes an enzyme which has at least one of the desired properties. As used herein, the terms xe2x80x9cisolated and/or xe2x80x9cpurifiedxe2x80x9d refer to in vitro isolation of a RNA, DNA or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or polypeptide, e.g., so that it can be sequenced, replicated, and/or expressed.